CN115724956A

CN115724956A - RBD-targeted high-neutralization-activity anti-SARS-CoV-2 fully-humanized monoclonal antibody 7B3 and application thereof

Info

Publication number: CN115724956A
Application number: CN202211149595.8A
Authority: CN
Inventors: 陈宇; 蓝柯; 吴莹; 刘乾运; 刘芳
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2022-09-21
Filing date: 2022-09-21
Publication date: 2023-03-03

Abstract

The invention provides a RBD-targeting high-neutralization-activity anti-SARS-CoV-2 fully human monoclonal antibody 7B3 and application thereof, wherein the monoclonal antibody 7B3 comprises a heavy variable region and a light chain variable region, and the amino acid sequences of three complementarity determining regions of the heavy chain variable region are respectively as follows: GGTFS, IIPA LDRA and AR; the light chain variable region has three complementarity determining regions with amino acid sequences: QSVSSD, GAS and QQYNNF. The monoclonal antibody 7B3 has high-efficiency and specific SARS-COV-2 resisting neutralizing activity, high expression, full humanity and high stability, and may be used in preparing new coronavirus detecting product and medicine for preventing and treating new coronavirus diseases.

Description

RBD-targeted high-neutralization-activity anti-SARS-CoV-2 fully-humanized monoclonal antibody 7B3 and application thereof

Technical Field

The invention belongs to the fields of microbiology and immunology, and relates to a RBD-targeted high-neutralization-activity anti-SARS-CoV-2 fully human monoclonal antibody 7B3 and application thereof.

Background

Coronavirus is an enveloped positive-strand RNA virus widely existing in nature, can infect a plurality of species such as poultry, bat, human and the like, causes diseases such as gastrointestinal tract, liver, respiratory system, nervous system and the like, and has great threat to human and livestock. At present, seven kinds of coronavirus capable of infecting human are known, wherein four kinds of low pathogenicity HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 are distributed globally and are ubiquitous in human, only respiratory tract infection is usually caused, and clinical symptoms are mild, namely fever, watery nasal discharge, cough and the like. In addition, the present century has also developed the pandemic of three highly pathogenic human coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2. 2019 coronavirus (COVID-19), the pathogen SARS-CoV-2 has a close relationship with SARS-CoV in evolution, the clinical symptoms of the two have extremely high similarity, the early infection stage mainly comprises symptoms such as fever, dry cough, systemic debilitation and the like, the late infection stage easily develops respiratory failure and acute pneumonia with different degrees, and the population with low immunity and basic diseases has high fatality rate. SARS-CoV-2 has extremely strong transmission ability and pathogenicity. The SARS-CoV-2 virus particle is spherical or elliptic and has a diameter of about 80-160 nm. The outermost layer of the virus particle is a capsule membrane consisting of lipid bilayers, a plurality of spinous processes with expanded ends are arranged on the capsule membrane, and a core consisting of non-segment single-stranded positive-strand RNA and nucleocapsid protein (N) of the virus is arranged in the virus particle. The viral genome is about 30kb in size and encodes 29 proteins in total, including 16 nonstructural proteins (nsp 1-nsp 16), 4 structural proteins (S, E, M, N), and 9 accessory proteins (ORF 3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF 10). The envelope of the virus particle contains three different glycoproteins, namely spike protein (S), membrane protein (M) and vesicle membrane protein (E). The S protein is the main antigen protein of coronavirus, the structure is a spherical structure with the extending tail end of the envelope, and the S protein has the functions of mediating the combination of virus and host cell receptor and promoting the fusion process of the envelope and cell membrane of the virus.

Although various vaccines against new coronavirus have been marketed so far, the protective effect of the vaccine is also severely challenged with the continuous variation of the virus. In addition, the existing specific medicines for treating the coronavirus are relatively lack, so that the economic cost is high, nonspecific treatment is generally adopted clinically, serious complications are prevented, the serious morbidity and mortality are reduced, and the cure rate is improved. Therefore, the development of general new coronavirus vaccines and specific therapeutic drugs with high protection efficiency becomes the first task of global emergency scientific research and development.

Therefore, there is a need to develop a fully human monoclonal therapeutic antibody with good protective effect against COVID-19.

Disclosure of Invention

In order to solve the technical problem, the invention provides a RBD-targeting high-neutralization-activity anti-SARS-CoV-2 fully human monoclonal antibody 7B3 and application thereof, and the fully human monoclonal therapeutic antibody has good protection effect on COVID-19.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a RBD-targeting, high neutralizing activity anti-SARS-CoV-2 fully human monoclonal antibody 7B3, said monoclonal antibody 7B3 comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region having the amino acid sequences of three complementarity determining regions: GGTFS, IIPA LDRA and AR; the light chain variable region has three complementarity determining regions with amino acid sequences: QSVSSD, GAS and QQYNNF.

Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 7B3 is as shown in SEQ ID NO:1 is shown in the specification; the amino acid sequence of the variable region of the light chain of the monoclonal antibody 7B3 is shown as SEQ ID NO:3, respectively.

Further, the amino acid sequence of the heavy chain constant region of the monoclonal antibody 7B3 is shown as SEQ ID NO:5, the amino acid sequence of the light chain constant region of the monoclonal antibody 7B3 is shown as SEQ ID NO: shown at 7.

Further, the monoclonal antibody further comprises:

the monoclonal antibody has the same function obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence;

or a heavy chain variable region comprising an amino acid sequence having at least 80% homology to said heavy chain variable region; and a light chain variable region having an amino acid sequence with at least 80% homology to said light chain variable region;

or the N end and/or the C end of the monoclonal antibody is connected with a label to obtain the antibody.

In other embodiments, V _H And/or V _L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence described above. V having the sequence as described above _H And V _L V of region height (i.e., 80% or more) homology _H And V _L Antibodies to the regions can be obtained as follows: mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) encodes the polypeptide of seq id no: 1-6, and then detecting the retained function of the encoded altered antibody using the functional assay described herein.

In other embodiments, conversion of the variable region gene to a scFv gene may be used, once the encoded V is obtained _H And V _L Fragment DNA fragments, i.e., those DNA fragments which can be further manipulated by standard recombinant DNA techniques, e.g., conversion of the variable region gene to a full-length antibody chain gene, fab fragment gene or scFv gene.

In these operations, V is encoded _L Or V _H Is operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein means that two DNA segments are linked together such that the amino acid sequences encoded by the two DNA segments remain in reading frame.

In a second aspect of the invention, the invention provides a nucleic acid molecule encoding the monoclonal antibody, comprising a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

Further, the polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody 7B3 are respectively as set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively.

Further, the polynucleotide sequence encoding the heavy chain constant region of the monoclonal antibody 7B3 is as set forth in SEQ ID NO: 6. the polynucleotide sequence of the light chain constant region is shown as SEQ ID NO: shown in fig. 8.

In a third aspect of the invention, there is provided an expression vector comprising said nucleic acid, said expression vector being capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell.

The vector may be specifically a plasmid vector, a phage vector, a viral vector or a mammalian expression vector. Mammalian expression vectors are specifically employed in the present invention.

In a fourth aspect of the invention, there is provided an engineered bacterium or eukaryotic host cell comprising said expression vector.

In the fifth aspect of the invention, the application of the monoclonal antibody in preparing COVID-19 therapeutic drugs or new coronavirus detection products is provided.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

the RBD-targeted high-neutralization-activity anti-SARS-CoV-2 fully human monoclonal antibody 7B3 provided by the invention shows good neutralization protection effect on SARS-CoV-2 infected cells. The result of the invention shows that the antibody has wide application prospect in preparing COVID-19 therapeutic drugs. Specifically, the monoclonal antibodies disclosed herein also have the following technical advantages:

(1) The whole human source does not need to be humanized and modified in clinical application.

(2) High neutralizing activity, the half effective concentration (EC 50) of the monoclonal antibody 7B3 to SARS-CoV-2 wild type is 4.976ng/mL on SARS-COV-2 infected cell model; the half-effective concentration (EC 50) of SARS-CoV-2Delta strain (B.1.617.2) was 6.036ng/mL.

(3) The action mechanism is clear: the monoclonal antibody 7B3 is highly specifically combined with RBD, shows that the monoclonal antibody is targeted to a receptor binding region and exerts an antiviral effect by specifically blocking the binding of viruses and receptors.

(4) The stability is good: because the monoclonal antibody genes are from the same cell of the human body and are naturally paired, the half-life period of the IgG1 antibody in the human body is known to be 21-28 days, and theoretically, the disclosed monoclonal antibody has consistent half-life period in the human body.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a diagram of single cell sorting by flow cytometry;

FIG. 2 is a diagram showing the detection pattern of the monoclonal antibody variable region gene amplification automated nucleic acid electrophoresis apparatus;

FIG. 3 is a detection profile of denatured SDS-PAGE after purification by affinity chromatography;

FIG. 4 is a graph showing the binding activity of 7B3 and RBD protein as a function of concentration;

FIG. 5 is a graph of the EC50 assay of antibodies on a pseudoviral cell model; wherein, FIG. 5A shows the half effective concentration of monoclonal antibody 7B3 against SARS-CoV-2 wild type; FIG. 5B is the median effective concentration of monoclonal antibody 7B3 against SARS-CoV-2Delta strain (B.1.617.2);

FIG. 6 is a graph of the EC50 assay of antibodies on a true virus cell model, wherein the left panel of FIG. 6 is the median effective concentration of monoclonal antibody 7B3 against the SARS-CoV-2 wild-type; FIG. 6 shows the half-effective concentration of monoclonal antibody 7B3 against SARS-CoV-2Delta strain (B.1.617.2).

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

The monoclonal antibody of the present application, its preparation method and application effect will be described in detail below with reference to examples and experimental data.

Example 1 screening and preparation of human anti-SARS-CoV-2 monoclonal antibody

1. Preparing a 96-well plate: mu.l of RNase-free water plus 20U of RNase inhibitor per well, covering with a sealing plate membrane, and standing at 4 ℃ for later use

2. Preparing a sample:

(1) And (3) cell recovery: taking out the frozen PBMC cells separated from peripheral blood of a new coronary pneumonia restorer from-80 ℃, quickly placing in warm water at 37 ℃, centrifuging at 800rpm for 5min after the cells are melted, and pouring out the supernatant; resuspending in a flow tube with 2-3ml FPBS, balancing, centrifuging at 800rpm for 5min, and pouring out the supernatant; resuspend with 2-3ml FPBS, centrifuge, and pour the supernatant. Finally, 100. Mu.l of FPBS was added for resuspension, and 2. Mu.l of the suspension was diluted 10-fold for cell counting.

(2) Singly dyeing the tube: 7 tubes (FVS-780, CD3-BV510, CD4-BV510, CD8-BV510, CD19-PE, igD-BB700, CD20-BV421, CD 38-FITC), 3X 10 tubes per tube ⁶ Cells, dyes were added to the flow tubes containing the cells individually at the antibody concentrations recommended by the instructions, and each reaction volume was made up to 50. Mu.l with FPBS.

(3) Naked cell control: 1 tube, 3X 10 ⁶ One cell, made up to 50. Mu.l with FPBS

(4) Cells used for sorting: 1 tube, the number of cells was determined first, and the final concentration was 100. Mu.l system (1X 10) ⁶ cells) and adding FVS-780 and CD3-BV510. CD4-BV510, CD8-BV510, CD19-PE, igD-BB700, CD20-BV421, CD38-FITC and Biotin-S1 fluorochrome.

(5) The samples were incubated at 4 ℃ for 1h in the dark.

(6) 3ml FPBS was added to each tube, centrifuged at 800g for 5min at 4 ℃, the supernatant was decanted, and the wash was repeated twice.

(7) After resuspension with 400. Mu.l FPBS, the cell pellet was removed using a 40 μm cell sieve and stored at 4 ℃ in the dark for sorting.

3. Flow type separation: selecting CD19 ⁺ ，CD3 ^- ，CD4 ^- ，CD8 ^- ，IgD ^- ，CD38 ⁺ ，CD20 ^- ，S1 ⁺ The cells of (a) are sorted. The results of flow sorting are shown in FIG. 1, with lymphocytes selected first, then non-adherent single cells, then viable cells, and then CD19 ⁺ 、CD3 ^－、CD4 ^－、CD8 ^－ Then selecting IgD ^－ Mature B cells of (4), and finally selecting S1 ⁺ The mature B cells are the target cells.

4. Amplification of fully human monoclonal antibody variable region gene by single cell-PCR technology

4.1 reverse transcription PCR reference (QIAGEN, 210212), the procedure is briefly described as follows: 94 cells were sorted by flow cytometry. All of the following specific primers for each subtype of heavy chain (H), kappa light chain (Kappa chain, kappa), and Lamda light chain (Lamda chain, lambda) were added simultaneously to each reaction system (see Table 1 for primer sequences).

Primer:

H：5′L-VH 1、L-VH 3、L-VH 4/6，5′L-VH 5、Hu IgG-const-anti、3′CμCH1

κ：5′L Vκ1/2、5′L Vκ3、5′L Vκ4、3′Cκ543–566

λ：5′L Vλ1、5′L Vλ2、5′L Vλ3、5′L Vλ4/5、5′L Vλ6、5′L Vλ7、5′L Vλ8、3′Cλ

TABLE 1 reverse transcription PCR primers

The PCR reaction system comprises: 5 Xbuffer 6 u L, dNTP 1.2 u L, reverse transcriptase (Takara biotechnology limited, RR 036A) 1.2 u L, primer, template for single cell, water to make up to 30L.

The PCR reaction conditions were: reverse transcription is carried out for 30min at 50 ℃; subsequently, pre-denaturation at 95 ℃ was carried out for 15min, at 95 ℃ for 40s, at 55 ℃ for 30s, at 72 ℃ for 1min, for 40 cycles, and finally extension at 72 ℃ for 10min.

4.2 nested PCR

Taking 1ul of the reverse transcription product as a template, and carrying out PCR (polymerase chain reaction) to amplify variable regions of H, kappa and lambda: primers for amplifying the heavy chain variable region, kappa light chain variable region and lambda light chain variable region are shown in Table 2 below.

TABLE 2 nested PCR primers

Note: the single-dashed section is for fusion with the upstream segment, and the dashed-black section is for fusion with the downstream segment.

The PCR reaction system comprises: DNA polymerase mixture (kang century Biotechnology Ltd., CW 2849) 12.5. Mu.L, primers as above, template of reverse transcription product 1. Mu.L, water to 25. Mu.L.

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min, followed by 94 ℃ 30s,57 ℃ 30s,72 ℃ 45min,40 cycles, and finally extension at 72 ℃ for 10min.

4.3 automated nucleic acid electrophoresis apparatus for detection

A portion of the results of the automated nucleic acid electrophoresis are shown in FIG. 2, where only the 7B3 heavy and light chain variable region bands are shown.

5. Synthesis of polynucleotide sequences of variable sequences

The antibody variable region DNA sequence was synthesized from the above polynucleotide sequence by Soviet Temple Biotech, inc. The antibody heavy chain variable region and the antibody light chain variable region are preceded by a signal peptide sequence and a restriction site (5.

6. Construction of plasmid expression vectors

From the antibody heavy chain constant region DNA sequence and the light chain constant region DNA sequence, kinzzhi Biotech limited, suzhou, was entrusted with the addition of restriction sites (5 'GAGCTCGGTACC-3') in front of and at the end of the heavy chain and light chain constant region DNA sequences and the addition of restriction sites (5 '-GCTAGC-3') in the end of the sequences (the heavy chain constant region sequence is represented by SEQ ID NO:5, the DNA coding sequence is represented by SEQ ID NO:6, the Kappa type light chain constant region sequence is represented by SEQ ID NO:7 and the DNA coding sequence is represented by SEQ ID NO: 8). The pCAGGS no-load plasmid (purchased from Shanghai Yuanmu Biotechnology Co., ltd., product number P0165) and the DNA sequence of the antibody heavy chain constant region are subjected to enzyme digestion by using restriction endonucleases Nhe I and Sac I, and the plasmid vector containing the antibody heavy chain constant region can be obtained by connecting the enzyme digested plasmid and the DNA fragment of the antibody constant region by using T4 DNA ligase. And carrying out enzyme digestion on the DNA sequences of the pCAGGS no-load plasmid and the antibody light chain constant region by using restriction endonucleases Nhe I and Sac I, and connecting the enzyme-digested plasmid and the antibody constant region DNA fragment by using T4 DNA ligase to obtain the plasmid vector containing the antibody light chain constant region.

And (3) carrying out enzyme digestion on the constructed plasmid vector containing the antibody heavy chain constant region and the antibody heavy chain variable region synthesized in the step 5 by utilizing restriction enzymes EcoR I and Kpn I, and connecting enzyme digestion products to obtain the eukaryotic expression vector capable of expressing the 7B3 antibody heavy chain. And (3) carrying out enzyme digestion on the constructed plasmid vector containing the antibody light chain constant region and the antibody light chain variable region synthesized in the step 5 by using restriction enzymes EcoR I and Kpn I, and connecting the enzyme digestion product by using T4 DNA ligase to obtain the eukaryotic expression vector capable of expressing the 7B3 antibody light chain. The two vectors can be used for expressing the antibody by means of transfecting eukaryotic cells.

The amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain variable region of monoclonal antibody 7B3 are respectively shown in amino acid sequences at 35-39, 57-64 and 103-104 positions of SEQ ID NO. 1; the amino acid sequences of CDR1, CDR2 and CDR3 regions of the light chain variable region are respectively shown as amino acid sequences at 36-41, 59-61 and 98-103 of SEQ ID NO. 3. The details are shown in Table 3.

TABLE 3

7. Transient expression and affinity chromatography purification of monoclonal antibody

Using an Expi293 expression system, mixing 15ug of heavy chain and 15ug of light chain, transfecting Expi 293F cells, operating according to the instructions of transfection reagents (EZ Trans Plus, AC04L 011), harvesting the culture fluid after 5-6 days, centrifuging to obtain about 50ml supernatant, using 5ml of preloaded Protein a affinity chromatography column, balancing with 20mM PBS before loading, injecting after the conductance is shown to be at baseline, after loading, washing the column with 20mM PBS until the baseline is stable, eluting the target Protein with 0.1m ph3.0 glycine buffer, stopping collection after OD280 is close to the baseline, washing the column with at least 3 column volumes of 20mM PBS, and washing the column with 20% ethanol after the baseline is stable. The SDS-PAGE result of the affinity chromatography purified monoclonal antibody is shown in figure 3. As can be seen from FIG. 3, the monoclonal antibody 7B3 was successfully purified.

Example 2 analysis of binding Activity of human anti-SARS-CoV-2 monoclonal antibody 7B3 with RBD

1. Coating: the RBD antigen is diluted to the concentration of 3 mug/mL by using the coating solution, an enzyme label plate is coated, each hole is 100 mug L, and the RBD antigen is coated overnight at 4 ℃.

2. And (3) sealing: adding 300 mul PBST lotion into each hole, washing 3 times multiplied by 3 min/time; the liquid in the wells was patted out, 2% BSA was added at 250. Mu.L/well and blocked at 37 ℃ for 1h.

3. Sample incubation: adding 300 mul PBST lotion into each hole, washing 3 times multiplied by 3 min/time; the liquid in the wells was patted out, purified mAb diluted with PBS was added to the first well at 9ug/ml, serially diluted 3 times at 100. Mu.L/well and incubated at 37 ℃ for 2h.

4. And (3) secondary antibody incubation: washing for 5 times, and operating the same as the above; HRP goat anti-human FC secondary antibody (1, 20000 diluted), 100 μ L/well, incubated at 37 ℃ for 1h.

5. Color development: washing for 5 times, and the operation is the same as above; adding 100 mu L of TMB single-component developing solution into each hole, developing for 15min at 37 ℃, adding 50 mu L of stop solution into each hole to stop the reaction, and detecting the light absorption value at 450nm by using an enzyme-labeling instrument. Standard curves were drawn using Graph Pad nonlinear regression, four parameter fit, and the mab EC50 concentrations were calculated from the standard curves and dilution fold.

6. As a result: see fig. 4. In FIG. 4, the curve shows the results of the detection of 7B3 and RBD, showing that the specific binding exhibits a dose-response relationship, indicating that the mAb is specific for RBD.

Example 4 analysis of neutralizing Activity on SARS-CoV-2 pseudovirus cell model

1. Pseudovirus packaging: different pseudoviruses are packaged according to the spike protein (S protein) of the wild-type SARS-CoV-2 and SARS-CoV-2 mutant strains. The hACE2 gene was stably transfected into BHK-21 cells to construct hACE2-BHK-21 cells stably expressing human ACE 2. Vero-E6 cells were transfected with plasmids expressing the different S proteins by lipofectamine 2000 (Biosharp, BL 623B). After 24h, 5h of VSV-dG-fLuc (1X 10) was diluted with DMEM ⁶ % tissue culture infectious dose [ TCID50]/mL) were inoculated with the transfected cells, then supplemented with growth medium (DMEM, 10% fetal bovine serum) containing vsv-g monoclonal antibody (I1-hybridoma, culture supernatant, 1. After 24 hours, the supernatant containing SARS-CoV-2 pseudovirus was collected, centrifuged at 3000rpm for 10 minutes, aliquoted, and stored at 80 ℃ by freezing. TCID50 of pseudovirus BHK-21-hACE2 cells were infected by serial dilution and calculated according to the Reed-Muench method.

2. Antibody dilution: SARS-CoV-2 pseudovirus (3X 10) ⁵ TCID 50/well) was incubated with diluted serum on 96-well white plates at room temperature for 30min, and then with trypsin-treated BHK-21-hACE2 cells at 2X 10 ⁴ Density mix per well.

3. The reading of Luciferase was checked and analyzed: after 16h of incubation, the medium of infected cells was removed, the cells were lysed with 1 × Bright-Glo luciferase assay reagent (Promega), and chemiluminescence detection was performed using a SpectraMaxiD 3 multiwell luminometer (Molecular devices). 50% neutralization dilution titers (NT 50) were calculated using GraphPad Prism 7 software and fitted with a non-linear regression curve.

The results are shown in FIG. 5 (the abscissa indicates the antibody concentration and the ordinate indicates the neutralization effect% relative to the negative control group). As shown in FIG. 5, the half Effective Concentration (EC) of 7B3 against SARS-CoV-2 wild-type pseudovirus in the SARS-COV-2 pseudovirus model ₅₀ ) 1.759ng/mL;7B3 half-Effective Concentration (EC) against SARS-CoV-2Delta pseudovirus strain (B.1.617.2) ₅₀ ) It was 3.784ng/mL.

Example 5 analysis of neutralizing Activity on cell model when SARS-CoV-2 is infected

1. Vero E6 cells were digested with 0.25% trypsin and then diluted to 5X 10 with medium (DMEM +10% FBS) ⁵ cells/mL, inoculated into 24-well cell culture plates, inoculated in a volume of 0.5 mL/well, placed at 37 ℃,5% CO2 cell culture box cultured overnight.

2. On the day of the experiment, antibody medium DMEM +2 fbs was diluted from the initial concentration in 5-fold series and a blank 24-well plate was added in a volume of 300 μ L/well; mu.L of COVID-19 virus suspension (diluted with DMEM +2% FBS and added to 120 PFU/well) was then added to each well, mixed well and incubated in a cell culture incubator for 1h.

3. Discarding cell culture supernatant in a 24-well plate, and adding 500 mu L of virus-antibody mixed suspension after co-incubation into each well; survival controls (no virus and antibody) and death controls (virus only) were set separately and incubated in a CO2 cell incubator at 37 ℃ 5% for 2h. The medium was discarded, and a medium containing 1% methylcellulose was added to continue the culture.

4. After 72h, the cell culture supernatant is discarded, 500 microliter of crystal violet staining solution is added for staining for 30min at room temperature, the staining solution is discarded, 1 mL/hole pure water is added, and the washing is repeated for 3 times.

5. Abandoning the washing liquid, patting the water in the plate holes by using absorbent paper, counting and recording the number of virus plaques, and calculating the EC50 value of the antibody.

6. The protective effect of the monoclonal antibody on the cell model and the result of EC50 show that the half effective concentration (EC 50) of 7B3 to SARS-CoV-2 wild type on the SARS-COV-2 infected cell model is 4.976ng/mL; the half-effective concentration (EC 50) of 7B3 against SARS-CoV-2Delta strain (B.1.617.2) was 6.036ng/mL.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A high neutralizing activity anti-SARS-CoV-2 fully human monoclonal antibody 7B3 targeting RBD, wherein said monoclonal antibody 7B3 comprises a heavy chain variable region and a light chain variable region, said heavy chain variable region having the amino acid sequences of three complementarity determining regions: GGTFS, IIPA LDRA and AR; the light chain variable region has three complementarity determining regions with amino acid sequences as follows: QSVSSD, GAS and QQYNNF.

2. The fully human monoclonal antibody 7B3 with high neutralizing activity against SARS-CoV-2 and targeting RBD according to claim 1, wherein the amino acid sequence of the heavy chain variable region of said monoclonal antibody 7B3 is as shown in SEQ ID NO:1 is shown in the specification; the amino acid sequence of the variable region of the light chain of the monoclonal antibody 7B3 is shown as SEQ ID NO:3, respectively.

3. The fully human monoclonal antibody 7B3 with high neutralizing activity against SARS-CoV-2 and targeting RBD according to claim 1, wherein the amino acid sequence of the heavy chain constant region of said monoclonal antibody 7B3 is as shown in SEQ ID NO:5, the amino acid sequence of the light chain constant region of the monoclonal antibody 7B3 is shown as SEQ ID NO: shown at 7.

4. The RBD-targeting high neutralizing activity anti-SARS-CoV-2 fully human monoclonal antibody 7B3 according to claim 1, wherein said monoclonal antibody further comprises:

5. A nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-4, wherein said nucleic acid molecule comprises a nucleic acid molecule encoding said heavy chain variable region and a nucleic acid molecule encoding said light chain variable region.

6. The nucleic acid molecule of claim 5, wherein the polynucleotide sequences encoding the heavy chain variable region and the light chain variable region of monoclonal antibody 7B3 are set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively.

7. The nucleic acid molecule of claim 5, wherein the polynucleotide sequences encoding the heavy chain constant region of monoclonal antibody 7B3 are each as set forth in SEQ ID NO:6, the polynucleotide sequences of the light chain constant regions are all shown as SEQ ID NO: shown in fig. 8.

8. An expression vector comprising the nucleic acid molecule of any one of claims 5 to 7, wherein said expression vector is capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell.

9. An engineered bacterium or eukaryotic host cell comprising the expression vector of claim 8.

10. Use of the monoclonal antibody of any one of claims 1-4 in the manufacture of a medicament for treating COVID-19 or a novel coronavirus detection product.